Despite strong evidence that the tendency towards obesity in humans is inherited the genes contributing to this neuroendocrine predisposition are not known. It is the goal of this project to construct a large scale physical map of and ultimately isolate the single gene that is mutated in a classical mouse model of obesity, the db/db mouse. This is an extensive proposed project in which serendipity may play an important role. Nonetheless, several systematic and complementary approaches can be utilized to make a directed attempt at identifying this important gene. Two vector systems will be employed to clone mouse chromosome 4 DNA fragments derived from a mouse/hamster hybrid cell line. A cosmid library will be constructed to isolate DNA fragments 20-50 kilobases in size and a yeast artificial chromosome library employing a system recently developed at Washington University will be constructed to isolate DNA fragments 100-700 kilobases in size. From these contiguous clones a detailed large scale genetic map of the region surrounding the db mutation will be assembled utilizing pulsed field gel electrophoresis, indirect end-label mapping, and hybridization with a panel of random oligonucleotide probes. Subsequently, a systematic screening of the derived gene sequences for the db locus wll be undertaken. Three approaches will be used: 1) searching for small deletions in this cloned region of mouse chromosome 4, 2) looking for variant or absent mRNA sequences in tissues such as brain (specifically hypothalamus) which are possible sites of expression of an abnormal db encoded gene product, and 3) screening sequences from this cloned region for clusters of non-methylated CpG dinucleotides which have been found to occur at the sites of expressed genes.